Methods and compositions for prophylactic and therapeutic treatment of infections

ABSTRACT

A new class of soluble phosphorylated glucans is described as well as the process for making the same. According to one embodiment, the soluble phosphorylated glucan is derived from the yeast Saccharomyces cerevisiae. The soluble phosphorylated glucans are useful for prophylactic and therapeutic applications against neoplastic, bacteria, viral, fungal and parasitic diseases. The soluble phosphorylated glucans are used either alone or in combination with a known antimicrobial agent for prophylactic and therapeutic antimicrobial applications. Additionally, they may be administered as a non-toxic adjuvant, in combination with chermotherapy. The soluble phosphorylated glucans are also useful for stimulating macrophage cells, either in vivo or in vitro, to produce a cytotoxic/cyctostatic factor effective against cancer cells.

CROSS REFERENCE TO RELATED APPLICATION

This aspplication is a continuation-in-part application of copendingSer. No. 767,388 filed on Aug. 19, 1985.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the lnvention

2.1 Immunobiological Activity of Particulate Glucans

2.2 Immunobiological Activity of Particulate Glucans

2.3 Unsucessful Attempts to Solubilize Particulate Glucans

3. Summary of the Invention

4. Brief Description of the Figures

5. Detailed Description of the Invention

5.1. Process for Preparation of Soluble Phosphorylated Glucan

5.2. Characterization of Soluble Phosphorylated

5.2.1. Elemental Composition

5.2.2. Structural Configuration

5.2.2.1. Molecular Sieving

5.2.2.2. Nuclear Magnetic Resonance Spectroscopy

5.3. Non-toxicity, Non-pyrogenicity and Non-Immunogenicity of SolublePhosphorylated Glucan

5.3.1. Non-Toxicity

5.3.2. Non-Pyrogenicity

5.3.3. Non-Immounogenicity

5.4. Prophylaxis and Therapy of Infectious Diseases

5.5. Routes and Methods of Administration

6. Preparation of Soluble Phosphorylated Glucans

6.1. Preparation from Particulate Glucan Obtained from Saccharomyces

6.2. Preparation from Coriolus versicolor

6.3. Preparation from Sclerotium

7. Immunobiological Properties of Soluble Phosphorylated Glucans

7.1. Modification of Enhanced Susceptibility to Opportunistic Infectionsin Immuno-suppressed Animals

7.1.1. Enhanced Survival

7.1.2. Enhanced Resistance of Immuno-suppressed Mice to Escherichia coliInfection

7.2. Modification of Bacterial Diseases in Animals by In VivoAdministration of Soluble Phosphorylated Glucan

7.2.1. Modification of Staphylococcus aureus Induced Sepsis

7.2.2. Modification of Escherichia coli-Induced Peritonitis

7.2.2.1. Time Required for Protective Effect

7.2.2.2. Dose-Response

7.2.2.3. Effectiveness of Soluble Phosphorylated Glucans

7.3. Effect of Soluble Phosphorylated Glucan on Viral-Induced Hepatitis

7.4. Effect of Orally Administered Soluble Phosphorylated Glucan onCandida albicans Induced Sepsis

7.5. Enhancement of Macrophage Phagocytic Activity

7.6. Enhancement of Macrophage Secretory Activity

8. Synergistic Effect of Soluble Phosphorylated Glucan and an AntibioticAgainst E. coli Induced Infection

1. FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions and methods forprophylaxis and therapeutic treatment of infections. More particularly,the invention relates to compositions and methods for prophylactic andtherapeutic treatment of infectious diseases induced by a variety ofmicroorganisms utilizing a novel class of soluble phosphorylated glucansin which the poly-[beta-(1-3) glucopyranose] chains are phosphorylatedin varying degrees.

2. BACKGROUND OF THE INVENTION

The term "glucan" refers generically to a variety of naturally occurringhomopolysaccharides or polyglucoses, including polymers such ascellulose, amylose, glycogen, laminarians, starch, etc. Glucanencompasses branched and unbranched chains of glucose units linked by1-3, 1-4, and 1-6 glucosidic bonds that may be of either the alpha orbeta type.

As defined herein, "particulate glucan" designates a water-insolubleparticulate (about 1-3 microns) polyglucose such as that derived fromthe cell wall of the yeast Saccharomyces cerevisiae. Particulate glucanis macromolecular and comprises a closed chain of glucopyranose unitsunited by a series of beta-1-3 glucosidic linkages. (Hassid et al.,1941, J. Amer. Chem. Soc. 63: 295-298; Di Luzio et al., 1979, Int'l J.Cancer 24: 773-779). X-ray diffraction studies have demonstrated thatparticulate glucans exist in the form of triple-stranded helices. (Sarkoet al., 1983, Biochem. Soc. Trans. 11: 139-142).

2.1. IMMUNOBIOLOGICAL ACTIVITY OF PARTICULATE GLUCANS

Particulate glucan is a potent activator of the macrophage/monocyte cellseries, complement, as well as of B cell lymphocytes. Thus, particulateglucan has profound effects on both the reticuloendothelial and immunesystems.

Previous studies have demonstrated that in vivo administration ofparticulate glucan to a variety of experimental animals induces a numberof profound immunobiological responses, including the following: (1)enhanced proliferation of monocytes and macrophages (Deimann and Fahimi,1979, J. Exper. Med. 149: 883-897; Ashworth et al., 1963, Exper. Molec.Pathol., Supp. 1: 83-103); (2) enhanced macrophage phagocytic function(Riggi and Di Luzio, 1961, Am. J. Physiol. 200: 297-300); (3) enhancedmacrophage secretory activity (Barlin et al., 1981, in Heterogeneity ofMononuclear Phagocytes, Forster and Landy, eds., Academic Press, NewYork, pp. 243-252); (4) increased macrophage size (Patchen and Lotzova,1980, Exper. Hematol. 8:409-422); (5) enhanced macrophage adherence andchemotactic activity (Niskanen et al., 1978, Cancer Res. 38: 1406-1409);and (6) enhanced complement activation (Glovsky et al., 1983, J.Reticuloendothel. Soc. 33: 401-413). Increased cytolytic activityagainst tumor cells has been demonstrated in macrophages from animalsand man treated with particulate glucan in both in vivo (Mansell and DiLuzio, 1976, in "The Macrophage in Neoplasia", Fink, ed., AcademicPress, New York, pp. 227-243) and in vitro studies [Schultz et al., inImmune Modulation and Control of Neoplasia by Adjuvant Therapy, M. A.Chirigos, ed. Raven Press, New York, pp.241-48 1978)].

Stimulation of the reticuloendothelial system by in vivo administrationof particulate glucan leads to inhibition of allogenic or xenogenic bonemarrow graft acceptance in lethally irradiated animals. (See, e.g.Wooles and Di Luzio, 1964, Proc. Soc. Exper. Biol. Med. 115: 756-759).This finding denotes that glucan will induce host defense mechanismseven against normal cells if they are genetically different from thehost.

In addition to effects on reticuloendothelial and immune responses, invivo administration of particulate glucan has been demonstrated toenhance hemopoietic activity including granulopoiesis, monocytopoiesisand erythropoiesis leading to greater recovery from a lethal dose ofwhole body irradiation (Patchen, 1983, Surv. Immunol. Res. 2: 237-242).

A number of studies have indicated that in vivo administration ofparticulate glucan significantly modifies host resistance to a widevariety of infectious diseases induced by bacterial, fungal, viral andparasitic organisms. In particular, enhanced host resistance toinfection has been shown when animals are challenged by microorganismssuch as Eshericheria coli, Staphylococcus aureus, Francisellatularensis, Mycobacterium leprae, Streptococcus pneumoniae, Candidaalbicans, Sporotrichum schenckii, as well as viruses such as Venezuelanequine encephalomyelitis virus, Rift Valley fever virus, murinehepatitis virus, frog virus III, Herpes simplex I and II, and parasitessuch as Leishmania donovani (see review by Di Luzio, 1983, Trends inPharmacol. Sci. 4: 344-347 and references cited therein).

Extensive studies have indicated that particulate glucan has potentanti-tumor activity. For example, particulate glucan has been shown toinhibit tumor growth and prolong survival in four syngeneic murine tumormodels including adenocarcinoma BW 10232, anaplastic carcinoma 15091A,melanoma B16, and spontaneous lymphocytic leukemia BW5147 (Di Luzio etal, 1979, in Advances in Experimental Medicine and Biology, Vol. 121A:269-290).

To evaluate the cellular basis of the anti-tumor activity of particulateglucan, the anti-tumor cytotoxic activity of peritoneal macrophages,derived from control and particulate glucan-treated mice, was studied(Mansell and Di Luzio, 1976, in The Macrophage in Neoplasia, Fink, ed.Academic Press, New York, pp. 227-243). These studies indicated thatperitoneal macrophages from glucan-treated mice produced a significantcytotoxic response compared to normal macrophages. This observation hasbeen confirmed (See, e.g., Ba rlin et al. 1981, in Heterogenity ofMononuclear Phagocytes, Forster and Landy, eds., Academic Press, NewYork, pp. 243-252) and Chirigos et al., 1978, Cancer Res. 38:1085-1091).

Additionally in vitro studies using normal and tumor cells incubatedwith particulate glucan have demonstrated that glucan exerts a directcytostatic effect on sarcoma and melanoma cells and a proliferativeeffect on normal spleen and bone marrow cells (Williams et al., 1985,Hepatology, 5: 198-206). These studies indicate that glucan, whenadministered therapeutically, will (1) significantly inhibit hepaticmetastases; (2) inhibit the growth of the primary tumor; and (3) enhancesurvival, possibly by increased Kupffer cell tumoricidal activity aswell as by a direct cytostatic effect of such glucan on sarcoma cells.

Notwithstanding these biological properties, the adverse side effects ofparticulate glucans have made these compounds all but useless inclinical medicine.

2.2. ADVERSE SIDE EFFECTS OF PARTICULATE GLUCANS

When particulate glucan is administered in vivo to animals, a number ofsevere side effects have beccme apparant, the most notable being:

(1) formation of granuloma;

(2) development of hepatosplenomegaly;

(3) increased susceptibility to endotoxins;

(4) activation of complement (anaphylyotoxin);

(5) development of pulmonary granulomatous vasculitis;

(6) development of hypotension following intravenous administration; and

(7) development of microembolism when administered at highconcentrations.

Additionally, there is a relatively high degree of acute toxicityobserved when particulate glucan is administered in vivo. For example,following a single intravenous injection of an aqueous suspension ofparticulate glucan, 20% and 100% morality were observed in micereceiving glucan at 250 and 500 mg/kg body weight respectively.

Moreover, due to the particulate nature of the glucan preparation (1-3microns), it is difficult to administer via an intravenous route. By wayof illustration, one patient receiving particulate glucan requiredconstant supervision during intravenous (IV) administration, continuousshaking of the IV drip bottole being essential to maintain theparticulate glucan in suspension to avoid formation of emboli in thepatient.

Although slightly soluble neutral glucans are commerically available,these preparations are not suitable for intravenous administrationbecause the aqueous solutions have very high viscosity and, moreimportantly, because their use when administered to experimental animalshas inevitably been accompanied by considerable toxicity.

Lentinan, a high molecular weight and poorly soluble beta-1,3 andbeta-1,6 glucan obtained from Lentinus edodes, has been studiedfollowing intravenous administration to dogs. A variety of adverseclinical effects were observed following adminstration of lentinan(Ajinomoto Co. Inc., Tokyo, Japan) at doses of 2.0, 8.0 and 30 mg/kg/dayfor 5 weeks. Adverse effects included vomiting, erythema, discolorationof the sclera, and facial swelling. Circulatory collapse, unsteady gait,altered behavioral patterns, excessive salivation were alos seen inindividual beagles. At autopsy, congestion of the gastrointestinalmucosa was observed in animals treated with 2.0 or 8.0 mg/kg/day.Morphological changes of liver indicated intracytoplasmic material,possibly lentinan, accumulating in liver cells. One animal showedcirculatory collapse upon the first injection at 8.0 mg/kg. While he didrecover, the animal experienced repeated vomiting episodes with presenceof blood indicating hemorrhaging of the gastrointestinal tract. Anotheranimal appeared to show a marked allergic response, as demonstrated byerythema and subcutaneous swelling (edema) of the face. Autopsy findingsdemonstrated extensive edema of subcutaneous tissue, and congestion ofthe gastrointestinal tract with hemorrhaging. Macrophage cells showedaccumulation of material, possibly lentinan. (Chesterman et al.,1981,Toxicol. Lett. 9: 87-90)

Additional toxicity studies were performed in which a variety of dosesof lentinan ranging from 0.1 to 1.0 mg/kg/day were given intravenouslyto rats for 9 weeks. Toxicity was manifested by the development ofcutaneous lesions and discoloration of the ears suggestingthromboembolic events. (Cozens et al., 1981, Toxicol. Lett. 9: 55-64).

2.3. UNSUCCESSFUL ATTEMPTS TO SOLUBILIZE PARTICULATE GLUCANS

In view of these disadvantages of particulate beta-1,3 glucans for invivo administration, extensive studies were undertaken to develop asoluble beta-1,3 polyglucose which might be non toxic, induce nosignificant pathology, and yet retain significant immunobiologicalactivity.

A low molecular weight non-phosphorylated soluble glucan preparationprepared by formic acid hydroylsis of particulate glucan has been shownto have anti-tumor and anti-staphylococcal activity (Di Luzio et al.,1979, Internat'l J. Cancer 24: 773-779). Unfortunately, the low yieldand diversity of fractions obtained by this method made this preparationnon-useful for prophylactic and therapeutic applications. (see Di Luzio,1983, Trends in Pharmacological Sciences 4: 344-347).

Similarly, attempts to solubilize particulate glucan by the addition ofdimethylsulfoxide (DMSO) a "molecular relaxant" were also unsuccessful.It was thought the DMSO would "relax" the triple helical configurationof the glucan molecule. However, particulate glucan did not dissolve inthe presence of DMSO. All attempts to isolate a soluble glucan from theDMSO solution resulted in failure. Upon dilution of the DMSO-glucansolution with various aqueous media such as glucose or saline solutions,the particulate glucan was reformed. Following dilution of theDMSO-soluble glucan solution with saline, all animals: receivinginjections of these solutions died immediately upon injection due tohigh concentration of DMSO or the reformation of the particulate glucan.Upon precipitation of the glucan in DMSO solution by the additon ofethanol (100%), the precipitate was collected and lyophilized. When thislyophilized glucan was added to water, the particulate glucan reformed.

Attempts to convert the neutral glucan preparation of particulate glucanto a polar-charged preparation by the addition of phosphate or sulfategroups as well as by acetylation were also unsuccessful. Each of theseprocedures was conducted following the attempted solubilization ofparticulate glucan by DMSO and in each instance the particulate glucanwas reformed.

3. SUMMARY OF THE INVENTION

During an exhaustive investigation of methods by which thetriple-stranded helices of glucan might be "relaxed" sufficiently topermit reaction of each of the chains, it was found that whenparticulate glucan was dissolved in a highly polar solvent (such asDMSO) in the presence of a strong chaotropic agent (such as urea), theglucan is sufficiently structurally disrupted to allow phosphorylationof each of the single chains (or strands) such that the resultantphosphorylated glucan shows the substantially complete absence of thecharacteristic triple helical structure of particulate glucan. Removalof the resultant phosphorylated glucan shows it to be soluble in water,non-toxic, non-immunogenic, substantially non-pyrogenic and capable ofexerting profound immunobiological responses when administered in vivoto animals and humans.

Based on these discoveries, the invention provides a new class ofsoluble phosphorylated glucans (a) in which thepoly-[beta-(1-3)glucopryanose] chains are phosphorylated in varyingdegrees; (b) which are non-toxic, non-immunogenic, substantiallynon-pyrogenic, and (c) which are capable of exerting pronouncedimmunobiological responses when administered in vivo in animals andhumans. These new soluble phopsphorylated glucans, which are furthercharacterized by a substantial absence of the triple helical structureof particulate glucans, immunostimulate macrophage activity withresulting activation of other immunoactive cells in thereticuloendothelial and immune systems. Additionally these solublephosphorylated glucans enhance hemopoietic activity including but notlimited to leukopoiesis. These soluble phosphorylated glucans exhibitcytostatic effects against adenocarcinomas and sarcomas in vivo, andagainst lymphocytic leukemia cells in vitro. Not only do these solublephosphorylated glucans stimulate macrophage cells in vivo, but theyexert profound stimulatory effects on macrophage cells cultured invitro. Such immunostimulation of macrophage cells is invariablyaccompanied by production of a macrophage cytotoxic/cytostatic factor(MCF), protein or proteins of unknown structure, which are selectivelytoxic to cancers cells, particularly adenocarcinomas.

Additionally, the invention provides a process for producing thesesoluble phosphorylated glucans by dissolving a particulate glucan(preferably prepared from Saccharomyces cerevisiae although othermicrobial sources may be used) in a highly polar solvent which containsa strong chaotropic agent, and reacting the resultant glucan withphosphoric acid to form a soluble phosphorylated glucan, and recoveringthe resultant phosphorylated glucans from the reaction mixture.

The invention provides compositions and methods for prophylaxis andtherapy of infections, induced by a variety of microorganisms includingbacteria, fungi, viruses and parasitic organisms, which compriseadministering an effective amount of a soluble phosphorylated glucan ora pharmaceutical composition comprising soluble phosphorylated glucan incombination with a physiologically acceptable carrier to an animal or ahuman. Additionally, compositions and methods are provided for therapyof infections induced by such agents which comprise administering atherapeutically effective amount of a composition comprising a solublephosphorylated glucan in combination with an antimicrobial agenteffective against the infection.

The immunobiological properties of the soluble phosphorylated glucansused in the present invention include (1) the ability to preventmortality due to overwhelming gram negative bacterial infections; (2)the ability to prevent mortality due to gram positive bacterialinfections; (3) the ability to modify mortality from spontaneousinfections in profoundly immuno-suppressed animals and man; (4) theability to modify enhanced susceptibility of immunosuppressed animalsand man to gram negative bacterial infections; (5) the ability tosignificantly modify viral infections; (6) the ability to modifyspontaneous infections induced by fungal and other parasiticmicroorganisms; (7) the ability to act synergistically withanti-microbial agents for the prophylactic and/or therapeutic treatmentof infections induced by microorganisms including bacteria, fungi,viruses and parasitic organisms and the like; (8) the ability tosignificantly inhibit primary tumor growth when used alone and to exerta synergistic effect against primary tumor growth when used incombination with anti-cancer agents; and (9) the ability to actsynergistically with anti-cancer agents in the regression of primarymalignant lesions as well as metastatic lesions in animals and man.

4. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, examples of specificembodiments and the appended figures in which:

FIG. 1 is a representation of the nuclear magnetic resonance spectrum ofsoluble phosphorylated glucan at 27 mg/ml.

FIG. 2 is representation of the nuclear magnetic resonance spectrum of acommercially available preparation of lentinan (Ajinomoto Co. Inc.,Tokyo, Japan). FIG. 2A is an illustration of the spectrum obtained at aconcentration of 40 mg/ml. FIG. 2B is an illustration of the spectrumobtained at a concentration of 3 mg/ml lentinan.

FIG. 3 is a graph illustrating the effect on survival ofcorticosteroid-immunosuppressed mice receiving twice weekly injectionsof soluble phosphorylated glucan. FIG. 3 also illustrates the effect ofchronic administration of soluble phosphorylated glucan on survival ofnormal mice.

FIG. 4 is a graph illustrating the effect of prior in vivoadministration of soluble phosphorylated glucan on resistance of bothnormal and corticosteroid-immunosuppressed mice to Escherichia coliinfection.

FIG. 5 is a graph showing the effect of prior treatment with solublephosphorylated glucan on the lethal effects of a subsequentexperimentally induced Staphylococcus aureus infection.

FIG. 6 is a graph illustrating the effect of prior treatment withsoluble phosphorylated glucan on survival of mice with subsequentexperimentally induced viral hepatitis.

FIG. 7 is a graph illustrating the effect of prior oral administrationof soluble phosphorylated glucan on survival of mice with experimentallyinduced Candida albicans infection.

FIG. 8 is a graph showing the effect of soluble phosphorylated glucan onInterleukin I production.

5. DETAILED DESCRIPTION OF THE INVENTION

Aqueous soluble phosphorylated glucan is advantageously used forprophylactic and/or therapeutic treatment of infectious diseases inducedby a variety of microorganisms. Aqueous soluble phosphorylated glucanrepresents a novel class of soluble phosphorylated glucans in whichpoly-[beta-(13-)glucopyranose]chains are phosphorylated in varyingdegrees. Soluble phosphorylated glucan shows the substantially completeabsence of the characteristic triple helical structure of particulateglucan.

Additionally, the novel soluble phosphorylated glucans are used incombination with a known antimicrobial agent for the treatment ofinfectious diseases.

Further, the soluble phosphorylated glucan of the present inventionprovides methods and compositions for treatment of malignant neoplasticdisease in animals and humans which comprise administering to an animalor a human a therapeutically effective amount of a solublephosphorylated glucan alone or in combination with an anti-cancer oranti-tumor agent. The invention also provides methods and compositionsfor prevention of leukopenia induced by administration of an anti-canceragent which comprise administering to an animal or a human, an effectiveamount of soluble phosphorylated glucan in combination with saidanti-cancer agent.

Furthermore, the invention provides methods for stimulating animal andhuman macrophage cells (in vivo or in vitro) to produce and secrete asoluble cytotoxic/cytostatic factor (MCF) and the product so produced.Specifically, MCF is produced by administering to an animal or a human asoluble phosphorylated glucan or by culturing animal or human macrophagecells in vitro in culture medium containing soluble phosphorylatedglucan. Copending application of Williams, Browder and DiLuzio, Ser. No.013,298 , filed on even date herewith is directed specifically tocompositions and methods for these applications and is incorporatedherein by reference.

5.1. PROCESS FOR PREPARATION OF SOLUBLE PHOSPHORYLATED GLUCAN

Aqueous soluble phosphorylated glucan is prepared by a process whichresults in a unique class of products different from any other glucanspreviously described.

Soluble phosphorylated glucan is prepared from particulate glucan, aneutral polyglucose derived, for example from Saccharomyces cerevisiae,as follows: particulate glucan is suspended in a solution of a strongchaotropic agent in an aprotic solvent such as dimethylsulfoxide (DMSO)with constant stirring. The strong chaotropic agent "relaxes" hydrogenbonding along the polyglucose chain, thus unfolding the molecule. It ispreferred to use a fairly high concentration of a strong chaotropicagent such as urea ranging from about 4-12 M to prevent reformation ofhydrogen bonds. The mixture is then heated and maintained at about50°-150° C. and phosphoric acid is slowly added with constant stirring.A precipitate comprising the soluble phosphorylated glucan product isapparent after about 1 hour. It is preferred to maintain the reactionmixture at about 100° C. for about 3-12 hours to increase the yield ofbioactive product. In practice, after reaction for about 6 hours atabout 100° C., the yield is approximately 70-90%. The degree ofphosphorylation of the soluble product varies slightly with reactiontime (e.g., 1.48% for 3 hours; 2.23% for 6 hours).

The bioactive soluble phosphorylated glucan product is isolated from thereaction mixture as follows: the mixture is cooled to stop thephosphorylation reaction and diluted with a volume of distilled watersufficient to resuspend the precipitate. The resulting solution isfiltered through coarse, medium and fine sintered funnels to remove anyremaining precipitate. The solution is then molecularly sieved to removeall components of less than about 10,000 daltons molecular weight (MW).Thus, DMSO, urea, glucose and any unreacted phosphoric acid are removedfrom the solution. Molecular sieving may be accomplished by any methodthat removes these low (i.e., less than about 10,000 daltons) MWcomponents. In one illustrative example, the solution is sieved usingSpectrapor membrane dialysis tubing and dialyzing against runningdistilled water for about 5 days. In another illustrative example, thesolution is sieved using a Millipore dialyzer/concentrator with a 10,000dalton MW membrane filter and a large volume of dialyzing solution.Following molecular sieving, the resulting solution is concentrated andlyophilized to yield the final soluble phosphorylated glucan in the formof a fluffy powder composition. Crystalline structures are not observed

The particulate glucan used in the process for preparing the solublephosphorylated glucan according to the present invention may be isolatedfrom the cell wall of S. cerevisiae by known methods (see e.g., Di Luzioet al., 1979, Internat'l J. Cancer 24: 773-779; Hassid et al., 1941, J.Amer. Chem. Soc. 63: 295-298 incorporated herein by reference). Briefly,in practice the particulate glucan is prepared as follows: dry yeast isdigested in aqueous sodium hydroxide solution and heated to about 100°C. for about 4 hours, then allowed to settle overnight. The supernatantis decanted and the procedure is repeated three times. The residue isacidified using hydrochloric acid, heated to and maintained at 100° C.for about 4 hours, and cooled overnight. The supernatant is decanted andthe acid digestion is repeated twice. The residue is then washedrepeatedly with distilled water and extracted with ethanol for at least24 hours. The reddish-brown supernatant is then aspirated and discarded.The ethanol extraction is repeated until the supernatant is essentiallycolorless. The ethanol is removed by repeatedly washing the residue withdistilled water. The particulate glucan is collected by centrifugationor filtration.

A variety of compounds, other than urea, known to function as "molecularrelaxants" were also evaluated to prevent reformation of hydrogen bondsafter DMSO had been used to "relax" the triple helical configuration ofparticulate glucan. These include (1) ethylene diamene tetracetic acid;(2) hydrazine sulfate; (3) monoethanol amine; (4) guanidine; (5)guanine, and (6) thiourea. Additionally, surfactants and emulsifyingagents such as Tween-20 and phospholipid emulsifying agents such asAlcolec and Centrolex f (lecithin) were also employed in an attempt tosolubilize and phoshorylate particulate glucan. In no case was a solubleimmunobiologically active preparation obtained.

Additionally, soluble phosphorylated glucan can be prepared from neutralpolyglucose or polyglucose-protein products derived from a variety ofother microbial sources. A non-exhaustive list of such sources ispresented in Table 1.

                  TABLE 1                                                         ______________________________________                                        EXAMPLES OF SOURCES OF GLUCAN WHICH CAN BE                                    EMPLOYED FOR THE PREPARATION OF SOLUBLE                                       PHOSPHORYLATED GLUCAN                                                         ______________________________________                                                 Alcaligenes faecalis                                                          Auricularia auricula-judae                                                    Auricularia polytricha                                                        Candida utilis                                                                Cladosporium fulvum                                                           Claviceps purpurea                                                            Cochiliobolus sativus                                                         Coriolus versicolor                                                           Corlinellus shiitake                                                          Corticium vagum                                                               Grifola umbellata                                                             Lentinus edodes                                                               Pichia fermentans                                                             Poria cocos                                                                   Saccharomyces cerevisiae                                                      Sclerotium coffeicolum                                                        Sclerotium delphnii                                                           Sclerotium glucanium                                                          Sclerotium rolsfi                                                             Shizophyllum commune                                                          Streptococcus salvarius                                                       Stereum sanguinolentum                                                        Wingea robertsii                                                     ______________________________________                                    

According to another alternate embodiment of the present invention, thesoluble phosphorlated glucan may be obtained from the medium used toculture an organism such as Sclerotium glucanium using a novel, rapidprocess. Briefly in practice, a colloidal glucan is prepared fromSclerotium glucanium as follows: a crude sclero-glucan, comprisirg apolyglucose chain of linearly arranged glucose units linked by beta 1-3glucosidic bonds in which about 30-35% of the linear chains have asingle glucose unit attached via a beta 1-6 bond obtained from themedium used to culture Sclecrotium glucanium, is mixed slowly withaqueous sodium hydroxide solution with heat and constant stirring. Themixture is allowed to stand at room temperature for about 4 days, heatedto about 50°-100° C. for about 15-60 minutes and then the mixture isallowed to cool to room temperature. The colloidal glucan is isolatedfrom the mixture either by centrifugation or filtration. To illustrate,when filtration is used a series of filters of decreasing pore size suchas 3.0, 1.2, 0.8, 0.65 microns are used. The resulting dark amberfiltrate is diluted with water and molecularly sieved to removed allcomponents of less than about 10,000 daltons molecular weight. In oneillustrative example, the mixture is sieved using spectropor membranedialysis tubing and dialyzing against 40 liters of water. The finalconcentrated mixture having about neutral pH is lyophilized yielding anamber spongy glucan material. In another illustrative example, themixture is dialyzed and concentrated using a Pellicon dialyzing unit.The mixture is dialyzed against 75 liters of pure water. The finalconcentrated mixture having about neutral pH is lyophilized yielding anamber spongy colloidal glucan material. Soluble phosphorylated glucan isprepared from the colloidal glucan as described above. Briefly,colloidal glucan is suspended in a solution of a strong chaotropic agentin an aprotic solvent such as dimethylsulfoxide (DMSO) with constantstirring. The strong chaotropic agent "relaxes" hydrogen bonding alongthe polyglucose chain, thus unfolding the molecule. It is preferred touse a fairly high concentration of a strong chaotropic agent such asurea ranging from about 4-12M to prevent reformation of hydrogen bonds.The mixture is then heated and maintained at about 50°-°150° C. andphosphoric acid is slowly added with constant stirring. A precipitatecomprising the soluble phosphorylated glucan product is apparent afterabout 1 hour. It is preferred to maintain the reaction mixture at about100° C. for about 3-12 hours to increase the yield of bioactive product.In practice, after reaction for about 6 hours at about 100° C., theyield is approximately 70-90%. The degree of phosphorylation of thesoluble product varies slightly with reaction time (e.g., 1.48% for 3hours; 2.23% for 6 hours).

5.2. CHARACTERIZATION OF SOLUBLE PHOSPHORYLATED GLUCAN

The solubility of the soluble phosphorylated glucan obtained from S.cerevisiae prepared according to the present invention is greater thanabout 50 mg/ml in water. Aqueous solutions of the soluble phosphorylatedglucan are non-viscous and do not taste sweet.

5.2.1. ELEMENTAL COMPOSITION

The elemental composition of the soluble glucan preparation, determinedby Galbraith Laboratories, (Knoxville, TN) is illustrated in Table 2.The data presented in Table 2 permits the average empirical formula ofthis preparation to be written as follows:

    C.sub.40 H.sub.87 PO.sub.37.

Thus, there is an average of one phosphate group for every 6.6 glucoseresidues in the soluble phosphorylated glucan.

                  TABLE 2                                                         ______________________________________                                        ELEMENTAL COMPOSITION OF                                                      SOLUBLE PHOSPHORYLATED GLUCAN.sup.a                                           ______________________________________                                        Element or Component                                                                             Mole %                                                     ______________________________________                                        Carbon             34.66                                                      Hydrogen           6.29                                                       Oxygen             42.83                                                      Nitrogen           0.64                                                       Sulfur             0.11                                                       Phosphorus         2.23                                                       Water of Hydration 11.78                                                      ______________________________________                                         .sup.a Determined after 6 hours phosphorylation.                         

5.2.2. STRUCTURAL CONFIGURATION

A number of methods were utilized to determine the molecular weight (MW)and various features of the structural configuration of the solublephosphorylated glucan.

5.2.2.1. MOLECULAR SIEVING

Column chromatography using Sepharose CL-6B-200 (Pharmacia FineChemicals, Piscataway, NJ) indicated that 80% of the soluble glucan hasa MW range from 10,000 to 100,000 daltons, while 20% has a MW range fromabout 100,000 to about 500,000 daltons.

5 2.2.2. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Carbon-13 nuclear magnetic resonance (¹³ C-NMR) spectroscopy using aBrucker WP-200 spectrometer (Brucker Instruments, Billerica, MA) wasperformed to determine several structural properties of the solublephosphorylated glucan from S. cervisiae prepared according to thepresent invention.

The samples for NMR studies were prepared using a 10% deuterium oxide(D₂ O) in water. Samples were first run with no reference material, andthen after the addition of a small aliquot of 1,4-dioxane. All sampleswere placed in 10 mm diameter tubes.

The ¹³ C-NMR spectral study of soluble phosphoryated glucan indicated abeta-1-3 glucan structure with no branching at the C-6 carbon (see FIG.1). The NMR spectrum indicates a substantial absence of the triplehelical structure of particulate glucans. The degree of phosphorylationwas estimated to be 3.6% which is in essential accord with analyticaldata.

In contrast to the spectra of soluble phosphorylated glucan from S.cerevisiae prepared according to the present invention, a lentinanpreparation (Ajinomoto Co. Inc., Tokyo Japan), a branched beta-1-3 and1-6 D glucan, at either 40 mg/ml (FIG. 2A) or 3 mg/ml (FIG. 2B)demonstrated attenuation of the NMR spectra. This is presumed to be dueto the gel state of this molecule, particularly at 40 mg/mlconcentration. No signals were obtained in a 10% D₂ O solution in thenon-gel 3 mg/ml concentration.

Comparison of FIG. 1 with FIG. 2 demonstrates complete structural andconformational differences between lentinan and soluble phosphorylatedglucan. In contrast to the disordered conformation of lentinan at thebeta-1-6 linkages (Saito et al., 1977, Carbohydrate Research, 58:293-305) ordered conformation of soluble phosphorylated glucan accordingto the present invention is manifested.

5.3. NON-TOXICITY, NON-PYROGENICITY NON-IMMUNO-GENICITY OF SOLUBLEPHOSPHORYLATED GLUCAN

Since the soluble phosphorylated glucan offers important advantages overparticulate glucan as an injectable biological response modulator,characteristic toxicity, pyrogenicity and immunogenicity of the solubleglucan are described below with particular reference to comparison ofthese properties of particulate glucan.

5.3.1 NON-TOXICITY

Acute toxicity was evaluated following a single intravenous injection ofsoluble phosphorylated glucan at a variety of doses into normal animals.Treated animals were observed for 30 days post-injection.

In one series of experiments, 49 ICR/HSD mice were divided into 3 groupsof 15 mice each and 2 groups of 2 mice each. Groups 1-3 received 0.5 mlsaline solution containing soluble phosphorylated glucan at respectively40, 200 and 1000 mg/kg; Groups 4 and 5, 1600 and 2000 mg/kg. Nomortality was observed in any group. Moreover, no physiological orbehaviorial alterations were apparent. In marked contrast, in micetreated similarly with particulate glucan, 20% mortality was observed at250 mg/kg and 100% mortality at 500 mg/kg.

In another series of experiments, two groups of 5 Sprague Dawley ratseach were treated with soluble phosphorylated glucan at respectively 250and 500 mg/kg via intravenous injection. No mortality or alteration ofphysiological or behavioral functions was apparent in either group. Incontrast, 30% and 100% mortality were observed following intravenousinjection of particulate glucan at 75 and 150 mg/kg respectively.

Chronic toxicity was evaluated following twice weekly intravenousinjections of saline solution containing soluble phosphorylated glucanat 0, 40, 200 and 1000 mg/kg doses. Body and organ weights, gross andmicroscopic pathology, serum electrolytes, solutes and serum enzymesindicative of renal and hepatic function were monitored.

In one series of experiments mice were weighed respectively at 0, 8, 11,15, 22 and 30 days post-treatment with soluble phosphorylated glucan. Nosignificant difference was observed in body weight at any dose ofsoluble phosphorylated glucan administered. After 30 days chronictreatment, animals were sacrificed. No change was seen in weight ofliver, lung and kidney. A statistically significant increase in spleenweight was noted in mice treated with 40 and 100 mg/kg soluble glucan(0.01<p<0.001), but not in mice treated with 200 mg/kg.

In another series of experiments, mice were weighed respectively at 0,15, 30, 49 and 60 days post-treatment (twice weekly) with solublephosphorylated glucan. No significant difference was observed in bodyweight at any dose of soluble phosphorylated glucan administered. After60 days chronic treatment with soluble phosphorylated glucan, animalswere sacrificed. No significant difference was observed in weight of theliver, kidney or lung. A statistically significant increase in spleenweight was apparent in mice treated with 1000 mg/kg solublephosphorylated glucan (p<0.00).

After 30 or 60 days chronic treatment, no significant alteration wasapparent in the following serum components: glucose, blood urea nitiogen(BUN), uric acid, cholesterol, triglycerides, total protein, albumin,globulin, creatinine, calcium, phosphorous, sodium, potassium, chloride,bicarbonate and anion gap. Moreover, no significant alteration wasapparent in the following enzymes: alkaline phosphatase, lacticdehydrogenase, serum glutamic oxalacetic transaminase, serum glutamicpyruvic transaminase and creatinine phosphokinase. No change wasdetectable in serum bilirubin.

Histological studies on tissues obtained from mice following 30 dayschronic treatment showed essentially normal liver histology in micereceiving 40 and 200 mg/kg soluble phosphorylated glucan per injection.In animals receiving 1000 mg/kg soluble phosphorylated glucan, monocyticinfiltrates were readily apparent in the liver. Lung and kidney tissueswere essentially normal in all mice.

Histological studies on tissues obtained from mice following 60 dayschronic treatment showed few hepatic granuloma of an isolated nature inanimals receiving injections at 40 and 200 mg/kg doses. A higher numberof monocytic infiltrates was observed in mice receiving injections at1000 mg/kg. In all autopsied animals, lung tissue was essentiallynormal.

Chronic toxicity was further evaluated using guinea pigs (Harlan SpragueDawley, Houston, Tex.) receiving 5 ml intraperitoneal injections ofsaline solution containing soluble phosphorylated glucan at 250 mg/kgfor 7 days (FDA required test). Results presented in Table 3 indicatethat there was an impairment of growth of guinea pigs receiving solubleglucan treatment when compared to controls receiving an equivalentvolume of 0.9% saline solution. Following 7 days chronic treatment, bodyweight of treated animals was, however, significantly increased by 9% ascompared to initial weight.

                                      TABLE 3                                     __________________________________________________________________________    EFFECT OF CHRONIC ADMINISTRATION OF                                           SOLUBLE PHOSPHORYLATED GLUCAN ON BODY WEIGHT                                  Mean Body Weight (gm).sup.a                                                   Days                                                                          Treatment                                                                           1     2     3     4     5     6     7                                   __________________________________________________________________________    Saline                                                                              213.8 ± 4.0                                                                      225.2 ± 6.6                                                                      227.4 ± 7.1                                                                      234.2 ± 7.3                                                                      239.7 ± 6.1                                                                      244.2 ± 5.2                                                                      253.8 ± 7.5                      SPGLN.sup.b                                                                         208.9 ± 2.9                                                                      202.3 ± 5.0                                                                      203.9 ± 5.3                                                                      207.4 ± 5.3                                                                      214.3 ± 6.0                                                                      215.6 ± 6.6                                                                      227.3* ± 6.6                     __________________________________________________________________________     .sup.a Values represent mean body weight (gm) ± standard error. N = 5      animals.                                                                      .sup.b SPGLN designates soluble phosphorylated glucan                         *p < 0.01                                                                

Chronic Toxicity was also evaluated in 2 adult female dogs receivingtwice weekly intravenous administration (5mg/kg) of solublephosphorylated glucan for 120 days. The dogs were fed Purina Chow andwater ad libitum supplemented with one can commercial dog food (Alpo™)twice weekly. Body weight and serum solutes, electrolytes and enzymeswere monitored at 0, 17, 24, 38, 80 and 120 days. Following 120 dayschronic treatment, a means weight gain of 2.8 kg or about 22% bodyweight was observed.

No significant difference was observed in the following serum solutes:glucose, BUN, uric acid, cholesterol, triglycerides, total protein,albumin, globulin, or creatinine. No significant difference was observedin the following serum electrolytes: calcium, phosphorous, sodium,potassium, chloride, bicarbonate, and anion gap. No significantdifference was observed in the following serum enzymes: alkalinephosphatase, lactic dehydrogenase, serum glutamic oxalacetictransaminase, serum glutamic pyruvic transaminase and creatininephosphokinase.

Additonally, no significant difference has been observed in the serumbiochemistry of a patient following therapy for 3 months with solublephosphorylated glucan at 50 mg/ml, administered three times per week.

5.3.2. NON-PYROGENICITY

Pyrogenicity of soluble phosphorylated glucan was evaluated following asingle intravenous injection to conscious dogs at doses of 7.5 mg/kg and30 mg/kg. Body temperature was monitored for 14 days post-injection.

Results presented in Table 4, demonstrate no pyrogenic reaction in thischronic animal model.

                  TABLE 4                                                         ______________________________________                                        ABSENCE OF AN ACUTE OR                                                        CHRONIC PYROGENIC RESPONSE IN DOGS                                                        Mean Body Temperature (°C.)                                Treatment Dose.sup.a                                                                      Time (Hours)                                                      (mg/kg)     0      1       6    24    144  336                                ______________________________________                                         7.5        38.3   37.4    38.3 38.3  38.3 38.4                               30.0        38.6   37.0    38.0 38.5  38.4 38.6                               ______________________________________                                         .sup.a N = 3 dogs/group.                                                 

Pyrogenicity was also evaluated using three dogs anesthetized withNembutal (30 mg/kg) receiving multiple injections of increasing doses of1, 5, 10, 15, 25 and 50 mg/kg of soluble phosphorylated glucan over athree hour period. Body temperature was determined at 15 minutesfollowing bolus injections. No pyrogenic effect was observed at anydose.

Pyrogenicity of soluble phosphorylated glucan was also evaluated inrabbits. Seven rabbits were divided into 2 groups of 2 and 5 rabbitseach. Group 1 received an isovolumetric saline solution; Group 2,received 5 mg/kg soluble phosphorylated glucan in saline solution byintravenous injection. Core body temperature was monitored at 15 minuteintervals for 100 minutes following a single bolus injection. Controlrabbits showed a mild decrease of 0.2.° C. in body temperature. Rabbitstreated with soluble phosphorylated glucan showed a mean increase of0.44.° C. Thus, there was a slight pyrogenicity seen in rabbits.

5.3.3. NON IMMUNOGENICITY

The interfacial ring test, designed to detect the presence of IgGantibodies, was used to evaluate the immunogenicity of solublephosphorylated glucan when chronically administered to dogs for 120days.

Serum samples were obtained from an adult female dog following 120 dayschronic treatment with twice weekly intravenous injections of 5 mg/kgsterile, pyrogen-free soluble phosphorylated glucan. The interfacialring precipitin test was performed as follows: 0.1 ml of undilutedantisera was pipetted into test tubes. The antigen or phosphorylatedsoluble glucan at dilutions of 1:2, 1:4, 1:8,.1:16 and 1:32 was layeredonto the anti-sera to form a straight interface. Formation of a whiteprecipitin ring at the interface, indicates that presence of antibodyspecific for the glucan. No precipitin ring was detected at any antigendilution. 5.4. PROPHYLAXIS AND THERAPY OF INFECTIOUS DISEASES

Due to the potent activity of the soluble phosphorylated glucan instimulating the immune response and reticuloendothelial system, it isadvantageously useful in prophylactic and therapeutic applicationsagainst diseases induced by a variety of microorganisms. Because solublephosphorylated glucan influences very fundamental host defense systemsof the body regulating the number, functional activity and interactionof macrophages, T and B lymphocytes, leukocytes and natural killer cellsas well as their humoral and secretory components, it possesses thepotential for non-specifically modifying an extensive array ofinfectious diseases.

Soluble phosphorylated glucan demonstrates a number of characteristicswhich make it particularly advantageous for the treatment of infectionsincluding, but not limited to the following advantages:

(1) Soluble phosphorylated glucan has a broad range of activity. It iseffective against infections induced by bacteria, fungi, viruses andparasitic organisms;

(2) Soluble phosphorylated glucan does not induce the development ofresistance in causative organisms because its effects are mediated bythe host;

(3) Soluble phosphorylated glucan has very low toxicity;

(4) Soluble phosphorylated glucan prevents and corrects the developmentof leukopenia;

(5) Soluble phosphorylated glucan enhances a variety of diverse aspectsof cellular and humoral immune responses of the host; and

(6) Soluble phosphorylated glucan prevents or reverses the developmentof immunosuppression in the host.

The soluble phosphorylated glucan is used to prevent and/or treatdiseases induced by gram positive bacteria including, but not limitedto: Staphylococcus aureus, Streptococcus pneumoniae, Haemophilusinfluenzae; gram negative bacteria including, but not limited to:Escherichia coli; Bacterium enteritis, Francisella tularensis; acid-fastbacteria including, but not limited to Mycobacterium tuberculosis, andMycobacterium leprae; viruses including but not limited to: Hepatitis;Herpes simplex I and II; etc.; fungi including but, not limited to:Candida albicans; Sporotrichum schenkii; and protozoal parasitesincluding but not limited to Leishmania donovani, Schistosoma mansoni,etc.

Additionally, the soluble phosphorylated glucan is used for theprevention and treatment of opportunistic infections in animals and manwhich are immunosuppressed as a result of either congential or acquiredimmunodeficiency or as a side-effect of chemotherapeutic treatment.

According to an alternate embodiment of the present invention, solublephosphorylated glucan is used advantageously in combination with a knownantimicrobial agent to prevent and/or treat diseases induced by grampositive bacteria including, but not limited to: Staphylococcus aureus,Streptococcus pneumoniae, Haemophilus influenzae; gram negative bacteriaincluding, but not limited to: Escherichia coli; Bacterium enteritis,Francisella tularensis; acid-fast bacteria including, but not limited toMycobacterium tuberculosis, and Mycobacterium leprae; viruses includingbut not limited to: Hepatitis; Herpes simplex I and II; etc.; fungiincluding, but not limited to: Candida albicans; Sporotrichum schenkii;and protozoal parasites including but not limited to Leishmaniadonovani, Schistosoma mansoni, etc.

Moreover, soluble phosphorylated glucan in combination with a knownantimicrobial agent is used for the prevention and treatment ofopportunistic infections in animals and man which are immunosuppressedas a result of congenital or acquired immunodeficiency or as aside-effect of chemotherapeutic treatment.

Soluble phosphorylated glucan has additive or synergistic effects whenused in combination with a broad range of antimicrobial agents effectiveagainst diseases induced by bacteria, fungi, viruses and parasiticorganisms and the like. Table 5 lists some of the antimicrobial agentsthat may be used according to the present invention in combination withsoluble phosphorylated glucan. Table 5 in no way is meant to be anexhaustive list. Moreover, a combination of one or more antimicrobialagents may be used together with soluble phosphorylated glucan.

                  TABLE 5                                                         ______________________________________                                        EXAMPLES OF ANTIMICROBIAL AGENTS                                              ______________________________________                                        I.   ANTIBACTERIALS                                                                Aminoglycosides                                                               Streptomycin                                                                  Neomycin                                                                      Kanamycin                                                                     Amikacin                                                                      Gentamicin                                                                    Tobramycin                                                                    Streptomycin B                                                                Dihydrostreptomycin                                                           Spectinomycin                                                                 Penicillin                                                                    Ampicillin                                                                    Hetacillin                                                                    Amoxicillin                                                                   Carbenicillin                                                                 Cephalosporins                                                                Cephaloridine                                                                 Cephalothin sodium                                                            Cephaloglycin dihydrate                                                       Cephalexin monohydrate                                                        Tetracycline                                                                  Tetracycline hydrochloride                                                    Oxytetracycline hydrochloride                                                 Chlorotetracycline hydrochloride                                              Doxocycline monohydrate                                                       Methacydine hydrochloride                                                     7-Chloro-6-dimethyltetracycline                                               Erythamycin                                                                   Sulfonamides                                                                  Carbomycin                                                                    Oleanodmycin                                                                  Troleandomycin                                                                Polymixin B collistin                                                         Chloramphenicol                                                          II.  ANTIFUNGALS                                                                   Amphotericin B                                                                Flucytosine                                                                   Nystatin                                                                      Grisiofulvin                                                                  Sulfamerizane                                                                 Thimerosal                                                                    Tolnaftate                                                               III. ANTIVIRALS                                                                    Acyclovir                                                                     3'-azido-3'-deoxythmyidine                                                    Vira A                                                                        Symmetrel                                                                     Idoxuridine                                                                   Bromovinyldeoxuridine                                                         (S)--9-(3-Hydroxy-2 phosphonyl methoxypropyl) adenine                    IV.  ANTIPARASITICS                                                                Sulfonamides                                                                  Pyrimethamine                                                                 Clindamycin                                                              ______________________________________                                    

Use of a combination of an antimicrobial agent together with solublephosphorylated glucan is particularly advantageous when theantimicrobial agent has toxic side effects. For example, antibacterialaminoglycosides such as gentamicin, streptomycin, and the like are knownto have serious toxicity, particularly ototoxicity and nephrotoxicity,which reduce the usefulness of such antimicrobial agents [see Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 6th ed., A.Goodman Gilman et al., eds; Macmillan Publishing Co., Inc., New York,pp. 1169-71 (1980)]. Use of soluble phosphorylated glucan in combinationwith such agents permits a lower dosage of the toxic antimicrobialagents while still achieving therapeutic effectiveness.

5.5. ROUTES AND METHODS OF ADMINISTRATION

The soluble phosphorylated glucans of the present invention can beadministered for prophylatic and therapeutic applications by a number ofroutes, including but not limited to: orally, by injection including butnot limited to intradermally, intraveneously, intraperitoneally,subcutaneously, intramuscularly, etc., topically as a dry powder or in asuitable physiologic carrier, by topical application to nasal andnasopharyngeal linings, and by inhalation via aerosolization andapplication to respiratory tract linings, etc.

When administered to an animal or a human, the soluble phosphorylatedglucan may be combined with water, an aqueous solution or anyphysiologically acceptable pharmaceutical carrier or vehicle. Moreoveras described above, the soluble phosphorylated glucan may beadministered in combination with an antimicrobial agent. When acombination of soluble phosphorylated glucan and an antimicrobial agentis desired, each agent may be simultaneously administered, or solublephosphorylated glucan may be combined with one or more antimicrobialagents and administered as a single composition. The combination may beadministered for prophylactic and therapeutic applications by a numberof routes, including but not limited to: orally, by injection includingbut not limited to intraveneously, intraperitoneally, subcutaneously,intramuscularly, intradermally, etc., topically as a dry powder or in asuitable physiologic carrier, by topical application to nasal andnasopharyngeal linings, and by inhalation via aerosolization andapplication to respiratory tract linings, etc.

According to other embodiments of the present invention, the solublephosphorylated glucan either alone or in combination with anantimicrobial agent may be administered by a number of deliverymodalities including but not limited to the following: incorporated intoliposome vesicles; in combination with an albumin complex to increasethe intravascular half-life, in combination with a targeting agent suchas a polyclonal or monoclonal antibody against a specific infectioninduced by agents such as a virus, bacterium, fungus or protozoalparasite, etc.; in combination with an anhydrous lipid base for topicalor parental administration, etc. In addition, the soluble phosphorylatedglucan, either alone or in combination with a known antimicrobial agentcan be impregnated on bandages, sutures or dressings either as a drypowder or in a suitable physiologic carrier.

The following series of Examples are presented for purposes ofillustration and not by way of limitation on the scope of the invention.

6. PREPARATION OF SOLUBLE PHOSPHORYLATED GLUCANS 6.1. PREPARATION FROMPARTICULATE GLUCAN OBTAINED FROM SACCHAROMYCES

Particulate glucan was prepared from Saccharomyces cerevisiae accordingto the method of Di Luzio et al. (1979, Int'l J. Cancer 24: 773-779).Briefly, using a 6 l flask, 540 gm of dry yeast (Universal Foods Corp.,Milwaukee, Wis.) was suspended in 3 l of 3% aqueous sodium hydroxidesolution. The suspension was placed in boiling water bath 4 hours,cooled overnight and the supernatant decanted. This procedure wasrepeated three times. The residue was then acidified with 800 ml ofconcentrated hydrochloric acid plus 2 l of 3% hydrochloric acid andplaced in a boiling water bath for 4 hours. The suspension was allowedto stand overnight and the supernatant decanted. The residue was furtherdigested with 3 l of 3% hydrochloric acid at 100° C. for 4 hours, cooledovernight and decanted. The 3% hydrochloric acid digestion was repeatedtwice. The residue was then washed three times with distilled water (20+C.) and twice with distilled water at 100° C. One l of ethyl alcohol wasadded to the residue, mixed thoroughly and allowed to stand a minimum of24 hours for maximum extraction. The dark reddish-brown alcoholsupernatant was aspirated from the residue and discarded. The alcoholextraction procedure was repeated until the alcohol supernatant wasessentially colorless. The alcohol was removed by washing the residuefour times with hot water; the particulate glucan preparation was thencollected by centrifugation, frozen and lyophilized.

Soluble phosphorylated glucan was prepared according to the methoddescribed in Section 5 by solubilization and phosphorylation of theparticulate glucan as follows:

18 gm of urea (8 M) was dissolved in a flask containing 50 mldimethylsulfoxide (DMSO) with constant stirring. One gm of particulateglucan was added to form a finely divided suspension. The flask washeated to 100° C. and 10 ml of phosphoric acid (85%) was added slowlydropwise. The mixture was maintained at 100° C. for 3-12 hours byimmersion in a boiling water bath. It is preferred to allow the reactionto proceed for about 6 hours.

During the heating process, a precipitate was formed which becamevisible after about 1 hour and increased in amount thereafter. Afterabout 6 hours, the mixture was cooled and diluted with 200 ml distilledwater to resuspend the precipitate. The mixture was then filteredthrough coarse, medium and fine sintered funnels to remove theprecipitate.

The resulting solution was then molecularly sieved in order to removelow molecular weight (MW) fractions including glucose, DMSO and urea.

In one series of experiments, molecular sieving was accomplished bydialysis for 5 days against running distilled water using Spectrapormembrane dialysis tubing (Fisher Scientific Co.; Pittsburgh, Pa.). TheMW size range of pores of this tubing is about 12,000 daltons. Inanother series of experiments, molecular sieving was accomplished usinga Millipore dialyzer/concentrator (Millipore Corp., Bedford, Mass.) witha 10,000 MW membrane filter. About 70 1 of dialyzing solution were usedto low MW compounds. In either case, tests for the presence of glucosein the final preparation were negative. Moreover, using high performancegas-liquid chromatography, no DMSO was detectable in the finalpreparation.

Following molecular sieving, the solution containing the phosphorylatedsoluble glucan was concentrated and lyophilized. This phosphorylatedglucan is stable in a lyophilized state for at least 2 years and atleast for 15 months in solution maintained at -20° C.

6.2. PREPARATION FROM CORIOLUS VERSICOLOR

Soluble phosphorylated glucan was prepared from a basidiomycete Coriolusversicolor (Fr. Quel.) as follows:

A commercially available polysaccharide-protein preparation termed"Krestin" or "PSK" obtained from Sanyko Corporation, Tokyo, Japan, wasused as the starting material for the preparation of solublephosphorylated glucan from C. versicolor. This commercial preparation ofa relatively crude preparation comprising a beta-1,4, beta-1,3,beta-1,6,-glucan-protein complex. The principal chemical structure ofthe polyglucose is a main chain of glucose units linked by 1-4glucosidic bonds, having attached branch or side chains of glucose unitslinked by beta-1,3 and beta-1,6 glucosidic bonds. The protein contentranges from 15-38%. (Ehrke et al., 1983, Internat'l J. Immunopharm. 534-42; Yamaura and Azuma, 1982, Adv. Immunopharm. 2: 501-507).

Because the commercially available PSK preparation is relatively crudepreparation and has a relatively high protein content, intraveousadministration of this material is not possible. It must be administeredorally.

The PSK polysaccharide-protein complex from C. versicolor was preparedas a soluble phosphorylated glucan (hereinafter "solublephosphorylated-PSK") as described in Section 5. The isolated solublephosphorylated-PSK was lyophilized.

6.3. PREPARATION FROM SCLEROTIUM

Soluble phosphorylated glucan was prepared from Sclerotium glucanium asfollows:

A commercially available sclero-glucan termed "Polytran R" obtained fromJetco Chemicals, Inc. (Corsicana, TX) was used as the starting material.This commercial preparation is a monionic polyglucose of greater than500,000 daltons MW comprising a linear chain of glucose units linked bybeta 1-3 glucosidic bonds in which about 30-35% of the linear chainshave appended a single glucose unit linked via a beta 1-6 linkage. Whenmixed with water or an aqueous solution, Polytran R forms a colloidalsuspension that becomes increasingly viscous over a 24 hour period.Because of the formation of such viscous gel, Polytran R is not usefulfor intravenous administration. A novel, rapid process was used toobtain a colloidal glucan from the sclero-glucan as follows:

One hundred forty grams Polytran R was added to 2.0 liters of 1 M NaOH,pH about 14 with constant stirring on a hot plate. The mixture wasmaintained at room temperature for 4 days. Then the mixture was heatedto about 85° C. for about 20 minutes, during which time a dark browncolor developed in the mixture. After the mixture was cooled to roomtemperature, it was filtered through coarse and then medium sinteredglass funnels. The filtrate was then passed serially through a series ofMillipore filters: 3.0, 1.2, 0.8 and 0.65 microns. The resulting clearfiltrate had a dark amber color. An aliquot (500 ml) was diluted to 6.0liters in a dialzing flask and dialyzed against 40 liters of ultrapurewater. The pH of the final concentrated mixture was about 6.7. Thecolloidal glucan isolated, a light brown spongy material, waslyophilized.

Soluble phosphorylated glucan was prepared from the colloidal glucan asdescribed in Section 5.

In practice, 72 gm of urea (6 M) was dissolved in a flask containing 200ml DMSO with constant stirring. Four gm of colloidal glucan was added toform a finely divided suspension. The flask was heated to 100° C. and 40ml of phosphoric acid (85%) was added slowly drop wise. The mixture wasmaintained at 100° C. for 6.0 hours.

During the heating process, a precipitate was formed which becamevisible and quite noticeable after about 3 hours and increased in amountthereafter. After about 6 hours, the mixture was cooled, and dilutedwith 4 liters distilled water to resuspend the precipitate. The mixturewas then filtered through a 3.0 micron filter.

The resulting solution was molecularly sieved in order to remove lowmolecular weight fractions. In one series of experiments molecularsieving was accomplished by dialysis (1 liter: 5 liters water).

Following molecular sieving, the solution containing the solublephosphorylate glucan was concentrated and lyophilized to yield about 4gms soluble phosphorylated glucan.

7. IMMUNOBIOLOGICAL PROPERTIES OF SOLUBLE PHOSPHORYLATED GLUCANS

In all experiments reported below, animals were maintained on PurinaLaboratory Chow ad libitum in air-conditioned rooms maintained on 12hour light/dark cycles.

7.1. MODIFICATION OF ENHANCED SUSCEPTIBILITY TO OPPORTUNISTIC INFECTIONSIN IMMUNOSUPPRESSED ANIMALS

Animals which are immunosuppressed either because of chemotherapy,congenital or acquired immunodeficiency (e.g., acquired immunodeficiencysyndrome or AIDS) are susceptible to infection by a variety ofopportunistic organisms. For example, a major cause of death in patientssuffering AIDS is pneumonia caused by Penumocystis carinii (see e.g.,Jaffee et al., 1983, J. Infect. Dis. 148:339-345; Gottlieb et al., 1981,New Eng. J. Med. 305:1425-1431).

Previous studies by Walzer et al. (1983, J. Reticuloendothel. Soc.33:1-9; 1979; Infec. Immunol. 24:939-947) have demonstrated that whenimmunosuppressed by chronic administration of a corticosteroid, theC3H/HeJ strain of mice is more susceptible to pneumonia induced by P.carinii than are other mouse strains. In these animals, like man,development of Pneumocystis pneumonia represents activation of latentinfection due to the development of immunosuppression.

The following experiments demonstrate that administration of solublephosphorylated glucan to chronically immunosuppressed C3H/HeJ micesignificantly enhanced survival and reduced susceptibility of theseanimals to opportunistic infections.

7.1.1. ENHANCED SURVIVAL

In one series of experiments, 100 C3H/HeJ mice (Jackson Laboratories,Bar Harbor, Me.) were divided into 4 groups of 25 mice each. Group 1received 0.5 ml of a 5% glucose solution intravenously; Group 2, 5 mgsoluble phosphorylated glucan intravenously; Group 3, 1.5 mg cortisoneacetate (Upjohn, Kalamazoo, Mich.) subcutaneously; and Group 4, both 5mg soluble phosphorylated glucan intravenously and 1.5 mg cortisoneacetate subcutaneously. All mice were treated twice weekly for 5 weeks,and survival was monitored for 60 days. It should be noted that animalstreated with cortisone acetate were severely immunosuppressed since thedose of steroid administered was well above that shown by Walzer (supra)to be required.

Results are illustrated in FIG. 3. As demonstrated in FIG. 3, there wasno mortality in mice treated either solely with glucose (Group 1) orsoluble phosphorylated glucan (Group 2). By day 24, survival of micetreated with corticosteroid alone was 12% (Group 3). Histopathologicalstudies indicated that contributing causes of death were bacterial andfungal infections, including those of the brain. In contrast, by day 24,survival in mice treated with corticosteroid and soluble phosphorylatedglucan was about 68% (Group 4). Long term survival of such nice was 66%,highly statisically significant when compared to corticosteroid-treatedmice (p<0.001).

7.1.2. ENHANCED RESISTANCE IMMUNO-SUPPRESSED MICE TO ESCHERICHIA COLIINFECTION

The following experiment demonstrates that administration of solublephosphorylated glucan enhanced resistance of both normal and chronicallyimmunosuppressed mice to infection by E. coli.

Sixty C3H/HeJ mice were divided into 4 groups of 15 mice each. Group 1recieved three injections of 5% glucose (0.5 ml) intravenously, Group 2,three injections of 4 mg/animal soluble phosphorylated glucanintravenously; Group 3, three injections of 1.5 mg/animal cortisoneacetate subcutaneously; and Group 4, three injections of a combined 4mg/animal soluble phosphorylated glucan intravenously and 1.5 mg/animalcortisone acetate subcutaneously at three day intervals. Three daysfollowing the last injection, the mice received 2.5×10⁷ E. coli bacteriaintraperitoneally. Survival was monitored for 15 days post-infectionwith E. coli.

Results illustrated in FIG. 4 demonstrate 65% survival in normalglucose-treated mice infected with E. coli (Group 1). No deaths occurredin this group after 7 days post-infection with E. coli. In contrast, 0%mortality was observed in normal mice treated with solublephosphorylated glucan (Group 2). Thus, pretreatment with solublephosphorlylated glucan significantly enhanced resistance to E. coliinfection in normal animals.

Results presented in FIG. 4 also demonstrate marked mortality of micechronically immunosuppressed by administration of corticosteroid. Inimmunosuppressed mice treated with glucose (Group 3), 50% mortality wasnoted at 40 hours and 75% mortality, at 5 days. In marked contrast, inimmunosuppressed mice treated with soluble phosphorylated glucan (Group4) 0% mortality was observed. Thus in both control and cortisone-treatedmice, soluble glucan was able to enhance host resistance resulting incomplete protection to a gram-negative infection both in normal andcortisone-immunosuppressed mice.

These studies, demonstrate that the administration of soluble glucan tomice receiving immunosuppressive doses of cortisone results inmodification of the cortisone-induced susceptibility to infection.Therefore, glucan is capable of modifying not only the immunosuppressiveeffects of chronic administration of cortisone leading to decreasedmortality from spontaneous infections, but also the relatively acuteimmunosuppressive effects of cortisone.

7.2. MODIFICATION OF BACTERIAL DISEASES IN ANIMALS BY IN VIVOADMINISTRATION OF SOLUBLE PHOSPHORYLATED GLUCAN 7.2.1. MODIFICATION OFSTAPHYLOCOCCUS AUREUS INDUCED SEPSIS

The following experiment demonstrates the effectiveness of in vivoadministration of soluble phosphorylated glucan in modifying the lethaleffects of sepsis associated with Staphylococcus aureus.

Twenty ICR/TEX mice were divided into two groups and treated as follows.At 3, 2 and 1 days prior to the induction of sepsis, Group 1 receivedintravenous injections of soluble phosphorylated glucan at 200 mg/kg;Group 2, designated a control group, received intravenous injections ofan equivalent volume of isotonic glucose. One day following the thirdadministration of polysaccharide, both groups received an intravenousinjection of 1.0×10⁹ cells S. aureus. Survival of experimental andcontrol mice was monitored for 130 days.

Results are illustrated in FIG. 5. As demonstrated therein, at 2 dayspost-infection with S. aureus, 60% mortality was observed in theglucose-treated mice (Group 2). At the same time, however, 0% mortalitywas observed in those animals treated with soluble phosphorylated glucanprior to infection. At 8 days post-infection, 27% of the control groupsurvived. In contrast, survival of the animals treated with solublephosphorylated glucan was 100% at the same time. No further deathsoccurred in either groups in 130 days. Thus, pre-treatment with solublephosphorylated glucan significantly reduced lethality of a subsequent S.aureus infection.

7.2.2. MODIFICATION OF ESCHERICHIA COLI INDUCED PERITONITIS 7.2.2.1.TIME REQUIRED FOR PROTECTIVE EFFECT

The following experiments demonstrate the effectiveness ofintraperitoneal administration of soluble phosphorylated glucan againsta subsequent experimentally induced E. coli sepsis.

Seventy nine adult white mice were divided into 8 groups of 8-13 animalseach. Group 1, designated control, received 1 ml glucose (5%); Group 2,designated positive control, 3 mg particulate glucan; and Group 3, 12.5mg soluble phosphorylated glucan at 24 hours prior to challenge with E.coli. Groups 4, 5, 6 and 7 received 12.5 mg soluble phosphorylatedglucan at respectively 6, 2, 1, 0 hours prior to challenge with E. coli.Group 8 received 12.5 mg soluble phosphorylated glucan at 2 and 4 hoursfollowing challenge with E. coli. All treatments were administered viaintraperitoneal injections. E. coli (1.0×10⁸ cells) was also injectedvia an intraperitoneal route. Results are illustrated in Table 6.

                  TABLE 6                                                         ______________________________________                                        EFFECT OF TIMING OF ADMINISTRATION OF                                         SOLUBLE PHOSPHORYLATED GLUCAN ON                                              MORTALITY DUE TO E. COLI SEPSIS                                                                        % Survival                                                                    Time (Hours)                                         Group Number    Time     Post-Infection                                       Treatment.sup.a (Hours).sup.b                                                                          12     16   24   48.sup.c                            ______________________________________                                        1.  Glucose         -24      40    0    0    0                                2.  Particulate Glucan                                                                            -24      100  100  88   88                                3.  Soluble Phosphorylated                                                                        -24      80   80   70   70                                    Glucan                                                                    4.  Soluble Phosphorylated                                                                         -6      100  100  100  88                                    Glucan                                                                    5.  Soluble Phosphorylated                                                                         -2      20   20   20   20                                    Glucan                                                                    6.  Soluble Phosphorylated                                                                         -1      20   20   20   20                                    Glucan                                                                    7.  Soluble Phosphorylated                                                                          0      30    0    0    0                                    Glucan                                                                    8.  Soluble Phosphorylated                                                                        +2, +4   80    0    0    0                                    Glucan                                                                    ______________________________________                                         .sup.a Group number refers to treatment protocol explained in text. The       number of animals in each group was 8-13.                                     .sup.b Time at which animals were treated.                                    .sup.c No additional mortality was observed in any group after the 48 hou     period.                                                                  

As demonstrated in Table 6, intraperitoneal administration of solublephosphorylated glucan signficantly enhanced survival of mice withexperimentally induce E. Coli peritonitis (Groups 3 and 4). As observed,the soluble phosphorylated glucan could effectively be administered aslate as 6 hours prior to challenge with E. coli.

Administration of soluble phosphorylated glucan either simultaneouslywith or subsequent to administration of E. coli was not effective inreversing the lethality of E. coli peritonitis (Groups 7 and 8) due tothe fulminant nature of the infection.

7.2.2.2. DOSE-RESPONSE

Another series of experiments was conducted to determine the effect ofadministration of various doses of soluble phosphorylated glucan on E.coli induced peritonitis and lethality.

One hundred and six white mice were divided into groups of 9-30 miceeach. Groups 1-2, designated controls received isovolumetric glucose.Groups 2-7 received soluble phosphorylated glucan at respectively 1, 1,2, 4, 8, 10, 20, 120, 200 and 260 mg/kg. All injections wereadministered intraperitoneally at 24 hours prior to the induction ofperitonitis by intraperitoneal administration of E. coli (1×10⁸ cells).Results are illustrated in Table 7.

                  TABLE 7                                                         ______________________________________                                        EFFECT OF DOSE OF SOLUBLE PHOSPHORYLATED                                      GLUCAN ON LETHALITY OF INTRAPERITONEAL OF                                     ADMINISTRATION OF E. COLI.sup.a                                               Group Number     Number   Dose      %                                         Treatment        of Mice  (mg/kg)   Survival                                  ______________________________________                                        1.  Glucose          30       --      13                                      2.  Soluble Phosphorylated                                                                         22       1       78                                          Glucan                                                                    3.  Soluble Phosphorylated                                                                         12       2       83                                          Glucan                                                                    4.  Soluble Phosphorylated                                                                         12       4       83                                          Glucan                                                                    5.  Soluble Phosphorylated                                                                         12       8       83                                          Glucan                                                                    6.  Soluble Phosphorylated                                                                          9       10      100                                         Glucan                                                                    7.  Soluble Phosphorylated                                                                          9       20      89                                          Glucan                                                                    ______________________________________                                         .sup.a Single injection of either glucose or soluble phosphorylated gluca     given intraperitoneally 24 hours before challenge with E. coli. Survival      was recorded at 24 hours postchallenge.                                  

As demonstrated in Table 7, all doses of soluble phosphorylated glucanranging from 1 mg/kg to 20 mg/kg were about equally effective inenhancing survival in animals challeged by intraperitoneal infectionwith E. coli.

7.2.2.3. EFFECTIVENESS OF SOLUBLE PHOSPHORYLATED GLUCAN

The following experiments further demonstrate the effectiveness ofintraperitoneal administration of soluble phosphorylated glucan againsta subsequently induced E. coli peritonitis.

In one series of experiments, the effectiveness of solublephosphorylated-PSK was demonstrated. Forty eight adult white mice weredivided into 3 groups of 16 animals each. Group 1 designated control,received 0.5 ml of isotonic saline solution intravenously; Group 2, 5 mgcommercially available PSK (Sankyo Corporation, Tokyo, Japan)intravenously; and Group 3, 5 mg soluble phosphorylated-PSKintravenously. All glucans were administered at 24 hours prior tochallenge with E. coli. E. coli (1×10⁸ bacteria) was injected via anintraperitoneal route. Results are illustrated in Table 8.

                  TABLE 8                                                         ______________________________________                                        EFFECT OF ADMINISTRATION OF SOLUBLE                                           PHOSPHORYLATED GLUCAN FROM C. VERSICOLOR                                      ON MORTALITY DUE TO E. COLI SEPSIS                                                           % Survival                                                                    Time (Hours) Post-Infection                                    Group.sup.a                                                                          Treatment     24       48     72                                       ______________________________________                                        1      Saline.sup.b   6        6      6                                       2      PSK.sup.c     63       25     19                                       3      Soluble Phos- 100      100    100                                             phorylated-PSK.sup.b                                                   ______________________________________                                         .sup.a N = 16 mice/group                                                      .sup.b Single injection of either saline or soluble phosphorylatedPSK (5      mg/mouse) was given intraperitoneally 24 hours before challenge with E.       coli.                                                                         .sup.c Commercially available PSK (Sankyo Corporation, Tokyo, Japan) was      administered intravenously (5 mg/animal) 24 hours before challenge with E     coli.                                                                    

As illustrated in Table 8, both the commercially available PSK and thesoluble phosphorylated-PSK glucans demonstrated significant protectiveactivity against E. Coli sepsis at 24 hours post-infection (i.e., 63%and 100% survival as compared to 6% in saline control animals Group 1).In marked contrast to the PSK preparation, however, the solublephosphorylated glucan-PSK prepared acording to the present inventionshowed greatly enhanced effectiveness against infection on a long termbasis. At 72 hours post-infection, no mortality was observed in thegroup treated with soluble phosphorylated-PSK (Group 3). At the sametime, 81% mortality was observed in the group treated with PSK (Group2).

In another series of experiments, the effectiveness of solublephosphorylated glucan obtained from Sclerotium glucanium wasdemonstrated.

Forty adult white mice were divided into 4 groups of 10 animals each.Group 1 designated control, received isovolumetric dextrose 5% (w/v);Group 2, 1 mg/mouse particulate glucan; Group 3, 1 mg/mouse solublephosphorylated glucan from Saccharomyces cerevisiae; and Group 4, 1mg/mouse SPG-RF, intraperitoneally. All control and glucan treatmentswere administered 24 hours prior to challenge with E. coli. E. coli(1×10⁸ bacteria) was injected via an intraperitoneal route. Results areillustrated in Table 9.

                  TABLE 9                                                         ______________________________________                                        EFFECT OF GLUCANS ON SURVIVAL OF                                              MICE WITH E. COLI SEPSIS                                                                 % Survival                                                                    Time (Hours) Post-Infection                                        Group.sup.a  14      24      48    72    96                                   ______________________________________                                        1.  Control       40%    0     0     0     0                                  2.  Particulate   80%    70%   70%   70%   70%                                    Glucan                                                                    3.  Soluble Phosphor-                                                                          100%    90%   90%   80%   80%                                    ylated Glucan.sup.b                                                       4.  Soluble Phosphor-                                                                          100%    100%  100%  100%  100%                                   ylated                                                                        Glucan-RF.sup.c                                                           ______________________________________                                         .sup.a Each group consisted of 10 animals.                                    .sup.b Soluble phosphorylated glucan obtained from Saccharomyces              cerevisiae.                                                                   .sup.c Soluble phosphorylated glucanRF represents soluble phosphorylated      glucan obtained from Scelerotium glucanium.                              

As illustrated in Table 9, all glucan preparations demonstratedsignificant protective activity against E. coli sepsis at 24 hourspost-infection (i.e., respectively 70%, 90%, 100% as compared to 0% inglucose control animals Group 1). The survival rate stabilized at 72hours post-infection. At that time, the Group 2 treated with particulateglucan showed only 70% survival. In contrast, however, animals treatedwith soluble phosphorylated glucan, Groups 3 and 4 showed 80% and 100%survival. Thus these preparations clearly are effective against E. coliinduced sepsis.

7.3. EFFECT OF SOLUBLE PHOSPHORYLATED GLUCAN ON VIRAL INDUCED HEPATITIS

Previous studies have demonstrated that particulate glucan is capable ofincreasing survival, inhibiting hepatic necrosis, and maintaining anactiviated state of phagocytic activity in mice challenged with murinehepatitis virus (MHV) strain A59 (Williams and Di Luzio, 1980, Science208:67-69).

The following experiment demonstrates that prior administration ofsoluble phosphorylated glucan enhanced survival of mice withexperimentally induced viral hepatitis.

Twenty male C57BL/6 Tex mice (Timco, Houston, Tex.) were divided intotwo groups. Group 1 designated controls, received intravenous injectionof glucose (0.5 ml/mouse) and Group 2 received intravenous injection ofsoluble phosphorylated glucan (5 mg/mouse) at 3, 2 and 1 days beforeinduction of acute viral hepatitis. Hepatitis was induced byintraperitoneal injection of 16 complement fixing units (CFU) of MHVstrain A59.

Survival of mice was monitored for 30 days following administration ofvirus. Results are illustrated in FIG. 6.

As demonstrated in FIG. 6, intravenous administration of solublephosphorylated glucan significantly enhanced survival in mice with acuteviral hepatitis. At 6 days post-induction of hepatitis, 50% survival wasobserved in the control group. In contrast, at that time 100% survivalwas observed in the group pre-treated with soluble phosphorylatedglucan. No further mortality was observed in either group (day 7-30).

7.4. EFFECT OF ORALLY ADMINISTERED SOLUBLE PHOSPHORYLATED GLUCAN ONCANDIDA ALBICANS-INDUCED SEPSIS

The following experiment demonstrates that orally administered solublephosphorylated glucan is effective in modifying the lethal effects ofsepsis induced by the yeast Candida albicans.

Twenty-five male ICR/Tex mice were divided into two groups. Group 1 (13animals) were maintained for seven days on drinking water containing 5.0mg/ml soluble phosphorylated glucan. The mice drink about 1.2-1.5ml/day, thus treated animals received about 7 mg/day solublephosphorylated glucan orally. Group 2, designated control group (12animals), were maintained on tap water throughout the experimentalperiod. On day zero, all animals were injected with Candida albicans(3.0×10⁶ cells/mouse) intravenously. Group 1 continued to receivesoluble phosphorylated glucan orally, in drinking water, for 5 dayspost-infection, then these animals were maintained on tap water. Thebody weight of mice in both groups was monitored. Mice in both groups 1and 2 gained weight at the same rate. Survival in both groups was alsomonitored. Results are graphically illustrated in FIG. 7.

As demonstrated in FIG. 7, oral administration of soluble phosphorylatedglucan pre-and for 5 days post-infection with C. albicans resulted in asignificant, but transient increase in survival of mice. At 25 and 30days post-infection, the survival in the soluble phosphorylatedglucan-treated group was significantly greater (at the p<0.05 level)than in the control group. The median survival time for the controlGroup was 18 days; whereas for the soluble phosphorylated glucan treatedgroup, median survival time was 30 days. With regard to long-termsurvival, however, the ultimate outcome was not significantly altered.Thus, this experiment suggests that higher dose of solublephosphorylated glucan may be needed when administered orally.

7.5. ENHANCEMENT OF MACROPHAGE PHAGOCYTIC ACTIVITY

The following experiment demonstrates that administration of solublephosphorylated glucan significantly enhanced phagocytic function ofmacrophages.

Twenty male ICR mice were divided into two groups of 10 each. At 3, 2and 1 day prior to determiniation of phagocytic activity, Group 1,designated control received injections of isovolumetric saline; Group 2,soluble phosphorylated glucan (200 mg/kg). All injections wereadministered intravenously.

Phagocytic function was evaluated by measuring the rate of intravascularclearance of colloidal carbon (C11/143 (a), Gunther Wagner, Hanover,Germany) according to the method of Wooles et al. (1962, Rad. Res. 16:546-554). Colloidal carbon was administered (640 mg/kg) intraveneouslyand serial blood samples were obtained from tail veins. Aliquots werehemolyzed in 4.0 ml of 0.5% sodium carbonate and the concentration ofcolloidal carbon was determined spectrophotometrically. The half-time(t/2) was taken as the time at which the optical density orconcentration was one-half the zero time value as determined byextrapolation of the clearance curves to zero time. Results arepresented in Table 10.

                  TABLE 10                                                        ______________________________________                                        EFFECT OF SOLUBLE PHOSPHORYLATED GLUCAN                                       ON MACROPHAGE PHAGOCYTIC FUNCTION                                                                               Intravascular                                          Body Weight                                                                              Liver Weight                                                                              Clearance                                   Treatment.sup.a                                                                          (gm)       (gm)        t/2 (minutes)                               ______________________________________                                        Saline     24.8 ± 0.82                                                                           1.94 ± 0.05                                                                            7.6 ± 0.73                               Soluble    25.2 ± 1.03                                                                           1.82 ± 1.03                                                                             3.5 ± 0.58*                             Phosphorylated                                                                Glucan                                                                        ______________________________________                                         .sup.a N = 10 per group.                                                      *p < 0.001                                                               

As demonstrated in Table 10, administration of soluble phosphorylatedglucan significantly enhanced phagocytic function as reflected by a 55%increase in clearance (Table 10). As previously observed, no increase inliver weight occurred (Table 10).

7.6. ENHANCEMENT OF MACROPHAGE SECRETORY ACTIVITY

A major secretory product of macrophages, particularly in an activatedstate, is a mediator molecule called Interleukin I. Interleukin I is apolypeptide which induces, both in experimental animals and man, anacute phase protein response. This response includes increasederythrocyte sedimentation rates, leukocytosis, elevated acute phaseproteins, and development of fever due to endogenous pyrogen.Additionally, Interleukin I has the ability to profoundly stimulateT-cell activation and proliferation. Thus, Interleukin I is a lymphocyteactivating factor capable of cellular recruitment and, hence, enhanceshost defense activity.

The following experiment demonstrates that administration of solublephosphorylated glucan stimulates macrophage secretory activity asassayed by the secretion of Interleukin I.

A number of rats (16) were injected intravenously with solublephosphorylated glucan (200 mg/kg). Eight control animals receivedisovolumetric glucose. Plasma samples were obtained at 1, 3, 5, 8, 24,48, 72 and 168 hours post-injection.

Production of Interleukin I was evaluated employing C-57 BL/6Jthymocytes. Cultures of thymocytes (1×10⁶ cells) were incubated withmedia alone, or with 0.1 ml plasma from control or solublephosphorylated glucan-treated rats. The cell cultures were maintainedfor 24 hours, at which time 1 uCi of ³ H-thymidine was added, andincubation was carried out for another 24 hour period. At that time,thymidine uptake was measured. Results are presented in FIG. 8.

As illustrated in FIG. 8, serum obtained from soluble phosphorylatedglucan-treated rats showed increased Interleukin I activity as reflectedby the elevation in thymidine uptake by thymocytes at 1, 24, 48 and 72hours compared to serum obtained from rats treated with glucose. Thus,soluble phosphorylated glucan rapidly activates macrophages andincreases secretory activity, as reflected by elevated plasmainterleukin levels. The nature of the 24-72 hour peak remains to beestablished.

8. SYNERGISTIC EFFECT OF SOLUBLE PHOSPHORYLATED GLUCAN AND AN ANTIBIOTICAGAINST E.COLI INDUCED INFECTION

The following experiments demonstrate the synergistic effectiveness ofin vivo administration of soluble phosphorylated glucan together with anantimicrobial agent against sepsis induced by E. coli.

Forty eight mice were divided into 4 groups of 12 mice each. Group 1designated control group received isotonic saline solution; Group 2,0.02 mg gentamicin intramuscularly; Group 3, 0.1 mg solublephosphorylated glucan intraperitoneally; and Group 4, a combination of0.02 mg gentamicin and 0.1 mg soluble phosphorylated glucan.

Animals treated with gentamicin either alone or in combination withsoluble phosphorylated glucan received the antimicrobial agent about 2hours prior to challenge with E. coli. Animals treated with solublephosphorylated glucan either alone or in combination with gentamicinreceived the glucan about 24 hours prior to challenge. All groups werechallenged by an intraperitoneal injection of a lethal dose of E. coli(1×10⁸ bacteria). Effect of the treatment on survival was monitored.Results are illustrated in Table 11.

                  TABLE 11                                                        ______________________________________                                        EFFECT OF SOLUBLE PHOSPHORYLATED GLUCAN                                       AND GENTAMICIN ON SURVIVAL FOLLOWING                                          SEPSIS INDUCED BY E. COLI                                                                      % Survival                                                   Treatment        Time (Hours) Post-Infection                                  Group No..sup.a  24       48       72                                         ______________________________________                                        1.  Control          18       4      0                                        2.  Soluble Phosphorylated                                                                         36       9      9                                            Glucan                                                                    3.  Gentamicin       14       9      0                                        4.  Soluble Phosphorylated                                                                         83       56*    52*                                          Glucan & Gentamicin                                                       ______________________________________                                         .sup.a N = 12 mice/group. Group number refers to treatment protocol           explained in text.                                                            *p < 0.05.                                                               

As demonstrated in Table 11, the control group showed 82%, 96% and 100%mortality at 24, 48 and 72 hours respectively. Group 2 treated withgentamicin along showed similar results, indicating that the doseadministered was ineffective against the lethal E. coli infection. Group3 treated with soluble phosphorylated glucan alone showed similarresults, also indicating that the dose of glucan was ineffective againstthe lethal E. coli infection.

In marked contrast, however, Group 4 treated with a combination ofsoluble phosphorylated glucan and gentamicin showed 83, 56 and 52%survival at 24, 48 and 72 hours respectively. Those animals which werealive at 72 hours, survived indefinitely. Hence these results clearlydemonstrate that combined administration of soluble phosphorylatedglucan and gentamicin exerted a synergistic effect leading to enhancedlong term survival to an LD₁₀₀ dose of E. coli.

In an effort to further evaluate the synergistic effect of the combinedtreatment, another group of mice were divided into 4 treatment groups.Group 1 designated control received isotonic saline solution; Group 2,0.02 mg gentamicin intramuscularly; Groups 3, 0.1 mg solublephosphorylated glucan intraperitoneally; and Group 4, a combination of0.02 mg gentamicin and 0.1 mg soluble phosphorylated glucan. All groupswere challenged by an intraperitoneal injection of a lethal dose of E.coli (1×10⁸ bacteria). Soluble phosphorylated glucan was administeredabout 24 hours prior to E. coli challenge. Gentamicin was administeredabout 2 hours prior to challenge with E. coli. The number of E. colipresent in the peritoneal cavity of animals from the various treatmentgroups was determined. Results are presented in Table 12.

                  TABLE 12                                                        ______________________________________                                        INFLUENCE OF SOLUBLE PHOSPHORYLATED                                           GLUCAN AND GENTAMICIN, ALONE OR IN                                            COMBINATION, ON E. COLI IN THE                                                PERITONEAL CAVITY                                                             Treatment       Concentration E. coli                                         Groups.sup.a    (No. cells/ml of Peritoneal Fluid)                            ______________________________________                                        1.  Control         483 × 10.sup.5                                      2.  Gentamicin      12 × 10.sup.5                                       3.  Soluble Phosphorylated                                                                        3 × 10.sup.5                                            Glucan                                                                    4.  Soluble Phosphorylated                                                                        1 × 10.sup.5                                            glucan + Gentamicin                                                       ______________________________________                                         .sup.a N = 8-14 mice/group.                                              

The concentration of viable E. coli in peritoneal fluid of control micewas 483×¹⁰ 5 /ml at 8 hours post-challenge (Group 1). This number is 41fold greater than that observed in the gentamicin-treated group whichwas about 12×10⁵ /ml (Group 2). Soluble phosphorylated glucan-treatedmice showed at 99.4% reduction in E. coli when compared to the salinegroup, indicating a marked effect of glucan alone.

When glucan and gentamicin treatments were combined, the antibacterialeffect was most pronounced. The number of bacteria in the combinedtherapy group was reduced by 99.8% when compared to the control, 92%when compared to the gentamicin-treated animals and 66% when compared tothe glucan treatment alone, respectively.

These studies, coupled with survival data, demonstrate the advantages ofcombined therapy. It is clearly possible to enhance the effectiveness ofantibiotics by administering glucan. This will allow employment of lowerdoses of antibiotics which may reduce toxicity.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

What is claimed is:
 1. A method for treatment of an infection in animalsor humans, comprising: administering to an animal or a human affectedwith said infection a therapeutically effective amount of a solubleglucan which comprises a phosphorylated poly-[beta-(1-3)glucopyranose]chain which is characterized by:(a) the capability of dissolving inwater or an aqueous solution; (b) being non-toxic, non-immunogenic andsubstantially non-pyrogenic; and (c) the capability of exerting apronouced immunobiological response when administered in vivo to ananimal or to a human.
 2. The method according to claim 1, in which thesoluble glucan is further characterized by the substantially completeabsence of triple helical chains as determined by nuclear magneticresonance spectroscopy.
 3. The method according to claim 1, in which thesoluble glucan is further characterized by having a degree ofphosphorylation ranging from about 1.4% to about 3.4%.
 4. The methodaccording to claim 1, in which the soluble glucan is furtherchracterized by having a molecular weight ranging from about 10,000 toabout 100,000 daltons.
 5. The method according to claim 1, in which thesoluble glucan is further characterized by having a molecular weightranging from about 100,000 to about 500,000 daltons.
 6. The methodaccording to claim 1, in which the soluble glucan is which is obtainedfrom a microbial source.
 7. The method according to claim 1, in whichthe infection is caused by a bacterium.
 8. The method according to claim7, in which the bacterium is selected from the group consisting ofStaphylococcus aureus, Streptococcus pneumoniae, Mycobacteriumtuberculosis, Hemophilus influenzae, Escherichia coli, Bacteriumenteritis, Francisella tularensis, and Mycobacterium leprae.
 9. Themethod according to claim 1, in which the infection is caused by avirus.
 10. The method according to claim 9, in which the virus comprisesHerpes simplex I or II or Hepatitis.
 11. The method according to claim1, in which the infection is caused by a fungus.
 12. The methodaccording to claim 11, in which the fungus comprises Candida albicans orSporotrichium schenkii.
 13. A method for the prevention of an infectionin animals or humans, comprising: administering to such an animal or ahuman a prophylactically effective amount of soluble glucan whichcomprises a phosphorylated poly-[beta-(1-3)glucopyranose] chain which ischaracterized by:(a) the capability of dissolving in water or an aqueoussolution; (b) being non-toxic, non-immunogenic and substantiallynon-pyrogenic; and (c) the capability of exerting a pronoucedimmunobiological response when administered in vivo to an animal or to ahuman.
 14. The method according to claim 13, in which the soluble glucanis further characterized by the substantially complete absence of triplehelical chains as determined by nuclear magnetic resonance spectroscopy.15. The method according to claim 13, which the soluble glucan isfurther characterized by having a degree of phosphorylation ranging fromabout 1.4% to about 3.4%.
 16. The method according to claim 13, in whichthe soluble glucan is further chracterized by having a molecular weightranging from about 10,000 to about 100,000 daltons.
 17. The methodaccording to claim 13, in which the soluble glucan is furthercharacterized by having a molecular weight ranging from about 100,000 toabout 500,000 daltons.
 18. The method according to claim 13, in whichthe soluble glucan is obtained from a microbial source.
 19. The methodaccording to claim 13, in which the infection is caused by a bacterium.20. The method according to claim 19 in which the bacterium is selectedfrom the group consisting of Staphylococcus aureus, Streptococcuspneumoniae, Mycobacterium tuberculosis, Hemophilus influenzae,Escherichia coli, Bacterium enteritis, Francisella tularensis, andMycobacterium leprae.
 21. The method according to claim 13, in which theinfection is caused by a virus.
 22. The method according to claim 21, inwhich the virus comprises Herpes simplex I or II or Hepatitis.
 23. Themethod according to claim 13, in which the infection is caused by afungus.
 24. The method according to claim 23, in which the funguscomprises Candida albicans or Sporotrichium schenkii.
 25. The methodaccording to claim 1, in which the glucan is administered intradermally,intravenously, intraperitoneally, subcutaneously, orally, topically,topically to nasal linings or by inhalation.
 26. The method according toclaim 13, in which the glucan is administered intradermally,intravenously, intraperitoneally, subcutaneously, orally, topically,topically to nasal linings or by inhalation.
 27. A method for treatmentof an infection in animals or humans, comprising: administering to ananimal or a human affected with said infection a therapeuticallyeffective amount of a composition which comprises a phosphorylatedpoly-[beta-(1-3)glucopyranose] chain which is characterized by:(a) thecapability of dissolving in water or an aqueous solution; (b) beingnon-toxic, non-immunogeric and substantially non-pyrogenic; and (c) thecapability of exerting a pronouced immunobiological response whenadministered in vivo to an animal or to a human, in combination with anantimicrobial agent effective against the infection.
 28. The methodaccording to claim 27, in which the composition is administeredintradermally intravenously, intraperitoneally subcutaneously, orally,topically, topically to nasal linings or by inhalation.
 29. A method forthe prevention or treatment of an opportunistic infection in animals orhumans which are immunosuppressed, comprising: administering to animmunosuppressed animal or human a prophylactically or therapeuticallyeffective amount of a souluble glucan which comprises a phosphorylatedpoly-[beta-(1-3)glucopyranose] chain which is characterized by:(a) thecapability of dissolving in water or an aqueous solution; (b) beingnon-toxic, non-immunogenic and substantially non-pyrogenic; and (c) thecapability of exerting a pronouced immunobiological response whenadministered in vivo to an animal or to a human.
 30. A pharmaceuticalcomposition for the prevention or treatment of an infection in animalsor humans, comprising: a therapeutically or prophylactically effectiveamount of a soluble glucan which comprises a phosphorylatedpoly-[beta-(1-3)glucopyranose] chain which is characterized by:(a) thecapability of dissolving in water or an aqueous solution; (b) beingnon-toxic, non-immunogenic and substantially non-pyrogenic; and (c) thecapability of exerting a pronouced immunobiological response whenadministered in vivo to an animal or to a human, and a physiologicallyacceptable carrier.
 31. The composition according to claim 30, furthercomprising an antimicrobial agent effective against the infection. 32.The composition according to claim 31, in which the antimicrobial agentis an aminoglycoside.
 33. The composition according to claim 32, inwhich the aminoglycoside is gentamicin.